A liquid chromatographic stationary phase containing immobilized membranes from cells expressing A2A adenosine receptor (A2AAR) is firstly described. Cellular membranes from CHO cells stably transfected with human A2AAR vector (A2A(+)) and from the same cell line transfected with the corresponding empty vector (A2A(−)) were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6 mm I.D. glass columns to create A2A(+)-IAM and A2A(−)-IAM stationary phases. Frontal chromatography experiments on both A2A(+)-IAM and A2A(−)-IAM columns demonstrated the presence of a low specific interaction with the receptor. However, immobilized A2A retained its ability to specifically bind known ligands as demonstrated by the agreement of the calculated Kd values with two different chromatographic protocols in comparison to previously reported data. In order to maximize the specific interaction, the same cellular membranes were immobilized on the inner surface of a silica capillary (40 cm×100 μm I.D.) by non-covalent interactions using the avidin–biotin coupling system to create two open tubular columns A2A(+)-OTand A2A(−)-OT. The open tubular system was characterized by ranking experiments for affinity studies in mixture useful for the selection of new potential candidates.
Development of new chromatographic tools based on A2Aadenosine receptor subtype for ligand characterizationand screening by FAC-MS
TEMPORINI, CATERINA;MASSOLINI, GABRIELLA;CALLERI, ENRICA
2013-01-01
Abstract
A liquid chromatographic stationary phase containing immobilized membranes from cells expressing A2A adenosine receptor (A2AAR) is firstly described. Cellular membranes from CHO cells stably transfected with human A2AAR vector (A2A(+)) and from the same cell line transfected with the corresponding empty vector (A2A(−)) were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6 mm I.D. glass columns to create A2A(+)-IAM and A2A(−)-IAM stationary phases. Frontal chromatography experiments on both A2A(+)-IAM and A2A(−)-IAM columns demonstrated the presence of a low specific interaction with the receptor. However, immobilized A2A retained its ability to specifically bind known ligands as demonstrated by the agreement of the calculated Kd values with two different chromatographic protocols in comparison to previously reported data. In order to maximize the specific interaction, the same cellular membranes were immobilized on the inner surface of a silica capillary (40 cm×100 μm I.D.) by non-covalent interactions using the avidin–biotin coupling system to create two open tubular columns A2A(+)-OTand A2A(−)-OT. The open tubular system was characterized by ranking experiments for affinity studies in mixture useful for the selection of new potential candidates.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
